目的： 探討支氣管上皮細(xì)胞與嗜酸性粒細(xì)胞聯(lián)合培養(yǎng)過程中炎性介質(zhì)的釋放及其機(jī)制. 方法：應(yīng)用流式細(xì)胞微珠實(shí)驗(yàn)(CBA)方法定量比較嗜酸性粒細(xì)胞、支氣管上皮細(xì)胞及其聯(lián)合培養(yǎng)上清液中單核細(xì)胞趨化蛋白(MCP)1的釋放以及p38 MAPK抑制劑SB 203580干預(yù)的影響;

　　【關(guān)鍵詞】支氣管,上皮細(xì)胞,嗜酸細(xì)胞,單核細(xì)胞化學(xué)吸引蛋白質(zhì)1,p38 MAPK

　　Regulatory role of p38 MAPK for MCP1 release from activated bronchial epithelial cells by eosinophils

　　【Abstract】 AIM: To investigate the release of monocyte chemoattractant protein (MCP)1 from the coculture of human bronchial epithelial cells and eosinophils and the related mechanisms. METHODS: MCP1 in culture supernatant was determined by Cytometric Bead Array (CBA) Kit in flow cytometry to compare MCP1 release in bronchial epithelial cells and eosinophils cultured alone or together, and investigate the inhibitive effect of SB 203580, a selective inhibitor of p38 MAPK, on MCP1 release. The reverse transcriptasepolymerase chain reaction (RTPCR) was used to analyze the gene expression of MCP1 in bronchial epithelial cells during coculture with eosinophils and the effect of SB 203580. RESULTS: The interaction of eosinophils and bronchial epithelial cells was found to upregulate the gene expression of MCP1 in epithelial cells. MCP1 in coculture supernatant of bronchial epithelial cells and eosinophils was elevated significantly [(1266±127) ng/L vs (134±25) ng/L,P<0.001]. Fixed eosinophils, which lost the ability to release MCP1, could also elevate epithelial cells to release MCP1 [(773±48) ng/L vs (107±15) ng/L, P<0.001] in coculture. SB 203580 could effectively inhibit MCP1 gene expression of bronchial epithelial cells during coculture with eosinophils, and decreased the release of MCP1 in the coculture of epithelial cells and eosinophils or fixed eosinophils [(1335±115) ng/L vs (481±42) ng/L, (868±89) ng/L vs (239±26) ng/L, P<0.001]. CONCLUSION: Eosinophils can activate bronchial epithelial cells to express MCP1 in coculture by p38 MAPK pathway so as to regulate the airway inflammatory reaction.

　　【Keywords】 bronchi; epithelial cells; eosinophils; monocyte chemoattractant protein1; p38 MAPK

　　【摘要】　用逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RTPCR)分析聯(lián)合培養(yǎng)過程中嗜酸性粒細(xì)胞對支氣管上皮細(xì)胞MCP1基因表達(dá)的活化及SB 203580對MCP1表達(dá)的抑制作用. 結(jié)果： 經(jīng)嗜酸性粒細(xì)胞活化后，支氣管上皮細(xì)胞中MCP1基因表達(dá)明顯上調(diào);嗜酸性粒細(xì)胞和支氣管上皮細(xì)胞聯(lián)合培養(yǎng)上清液中MCP1蛋白質(zhì)釋放顯著增加[(1266±127) ng/L vs (134±25) ng/L, P<0.001];多聚甲醛固定后的嗜酸性粒細(xì)胞不能釋放MCP1，但其在與支氣管上皮細(xì)胞聯(lián)合培養(yǎng)過程中仍可增加支氣管上皮細(xì)胞釋放MCP1 [(773±48) ng/L vs (107±15) ng/L, P<0.001];p38 MAPK抑制劑SB 203580可有效抑制嗜酸性粒細(xì)胞活化支氣管上皮細(xì)胞表達(dá)MCP1基因，顯著降低正常嗜酸性粒細(xì)胞和多聚甲醛固定嗜酸性粒細(xì)胞與支氣管上皮細(xì)胞聯(lián)合培養(yǎng)過程中MCP1的釋放[(1335±115) ng/L vs (481±42) ng/L和(868±89) ng/L vs (239±26) ng/L, P<0.001]. 結(jié)論： 嗜酸性粒細(xì)胞可通過p38 MAPK信號傳導(dǎo)通路活化支氣管上皮細(xì)胞表達(dá)MCP1，調(diào)控過敏性氣道炎癥反應(yīng).

　　0引言

　　過敏性哮喘的顯著特點(diǎn)就是大量炎性細(xì)胞，特別是嗜酸性粒細(xì)胞在支氣管炎癥部位聚積，活化后的嗜酸性粒細(xì)胞可通過脫顆粒釋放嗜酸性粒細(xì)胞陽性蛋白(ECP)、主要堿性蛋白(MBP)等毒性蛋白，而這些毒性蛋白可造成支氣管上皮損傷[1]. 損傷的支氣管上皮修復(fù)和氣道重建可導(dǎo)致氣道水腫、增厚、狹窄和對不同刺激的反應(yīng)性增高，從而引發(fā)常見的咳嗽、呼吸困難等一系列臨床癥狀. 支管上皮對外環(huán)境的屏障功能已被廣泛認(rèn)識，但作為哮喘炎癥反應(yīng)的發(fā)生部位，其在過敏性哮喘發(fā)生、發(fā)展中的免疫調(diào)控作用未能引起廣泛重視. 我們通過嗜酸粒細(xì)胞與支氣管上皮細(xì)胞接觸培養(yǎng)為實(shí)驗(yàn)?zāi)Ｐ停接懴^程中嗜酸粒細(xì)胞從外周血移行至支氣管上皮后，通過活化支氣管上皮釋放炎性介質(zhì)細(xì)胞趨化因子MCP1，調(diào)控過敏性哮喘炎性反應(yīng)的過程.

　　1材料和方法

　　1.1材料密度1.082 kg/L Percoll溶液: 混合87.92 mL Percoll原液(密度1.130, 瑞典Amersham公司產(chǎn)品)，15 mL 1.5 mol/L NaCl